Targeted genome modification using circular single-stranded DNA

Abstract

The present invention is directed to methods for generating one or more genetically modified cells by using a circular single stranded DNA (CiSSD) as a donor template and targeting genome modification. These methods include transferring one or more DNA polynucleotides into the cell for site-specific nuclease-mediated DNA repair and selecting one or more cells having the transferred DNA incorporated into the cell's genome.

What is claimed is: 
     
       1. A method for generating a genetically modified cells, comprising:
 transferring a circular single stranded DNA (CiSSD) having a DNA insert, a 5′ homology arm, and a 3′ homology arm to a cell, wherein the 5′ homology arm and the 3′ homology arm are complementary to the polynucleotides in a target region of a genomic DNA in the cell; 
 inducing a nucleotide break with Cas nuclease in the target region of genomic DNA in the cell; 
 hybridizing the 5′ homology arm and the 3′ homology arm of the CiSSD with the complementary polynucleotides in the target region of the genomic DNA; and 
 inserting the DNA insert into the target region of genomic DNA; 
 whereby a genetically modified cell is generated, and the CiSSD has a higher integration efficiency than that using a linear single stranded DNA under the same condition. 
 
     
     
       2. The method of  claim 1 , further comprising selecting one or more cells having the DNA insert. 
     
     
       3. The method of  claim 1 , wherein the nucleotide break is a double-stranded nucleotide break. 
     
     
       4. The method of  claim 1 , wherein the CiSSD further comprising an initiator sequence and a terminator sequence. 
     
     
       5. The method of  claim 1 , wherein the cell is a T cell or a natural killer cell. 
     
     
       6. The method of  claim 1 , wherein the DNA insert encodes a chimeric antigen receptor (CAR). 
     
     
       7. The method of  claim 6 , wherein the genetically modified cells are CAR-modified T cells. 
     
     
       8. The method of  claim 6 , wherein the genetically modified cells are CAR-modified natural killer cells. 
     
     
       9. The method of  claim 1 , wherein the genetically modified cells are non-human embryonic stem cells. 
     
     
       10. The method of  claim 1 , wherein the insert is about 2 kB to 20 kB in length. 
     
     
       11. The method of  claim 1 , wherein the insert is about 2 kB to 10 kB in length. 
     
     
       12. The method of  claim 1 , wherein the insert is about 1.6 kB to 5 kB in length. 
     
     
       13. The method of  claim 1 , wherein the 5′ homology arm and the 3′ homology arm are each between about 50 nucleotides to 3000 nucleotides in length. 
     
     
       14. The method of  claim 13 , wherein the 5′ homology arm and the 3′ homology arm are each about 300-500 nucleotides in length.